Plasma Membrane Ca2+ Release-Activated Ca2+ Channels With A High Selectivity for Ca2+ Identified by Patch–Clamp Recording in Rat Liver Cells

Abstract

Repetitive waves of increased cytoplasmic Ca²⁺ concentration play a central role in the process by which hormones regulate liver function. Maintenance of these Ca²⁺ waves requires Ca²⁺ inflow through store–operated Ca²⁺ channels. The properties and mechanism(s) of activation of these channels are not well understood. Store–operated Ca²⁺ channels (SOCs) in the H4–IIE rat liver cell line were studied by whole–cell patch clamping. Depletion of Ca²⁺ in intracellular stores by intracellular perfusion with either inositol 1,4,5–trisphosphate (InsP₃) or thapsigargin in the presence of 10 mmol/L ethylene glycol–bis(β–aminoethyl ether)–N,N–tetraacetic acid (EGTA), or with 10 mmol/L EGTA alone, activated an inward current that reversed at a membrane potential above +40 mV. In physiologic extracellular medium, this inward current was carried exclusively by Ca²⁺ and was blocked by a variety of di– and trivalent cations. In the absence of extracellular Ca²⁺ and Mg²⁺, the inward current was carried by monovalent cations. This current was 10 to 30 times larger than that observed in the presence of extracellular Ca²⁺, and permitted the detection of single–channel events that corresponded to a single–channel conductance of about 40 pS. Both the Ca²⁺ and Na⁺ inward currents were blocked by the calmodulin antagonist, N–(6–amino hexyl)–5–chloro–1–naphthalenesulphonamide (W7), but not by calmidazolium or calmodulin–dependent protein kinase II fragment 290–309. It is concluded that liver cells possess plasma membrane Ca²⁺ channels that have a high selectivity for Ca²⁺, are activated by a decrease in the concentration of Ca²⁺ in intracellular stores through a mechanism that is unlikely to involve calmodulin, and are involved in re–filling intracellular Ca²⁺ stores during Ca²⁺ signaling.